The development of a safe and effective HIV-1 vaccine is a global health priority. Plasmid DNA vaccines have elicited potent virus-specific cellular immune responses in animal models but have proven only marginally immunogenic in clinical trials to date. Strategies aimed at augmenting the immunogenicity of DNA vaccines are therefore an area of active investigation. We have recently demonstrated that co-administration of the combination of plasmid MIP-1alpha and plasmid GM-CSF with DNA vaccines resulted in the recruitment of large numbers of dendritic cells to the site of inoculation and a synergistic and durable augmentation of DNA vaccine-elicited immune responses in mice. We hypothesize that the immunogenicity of DNA vaccines is limited by the availability of professional antigen-presenting cells (APCs) at the site of inoculation. We further hypothesize that DNA vaccine-elicited immune responses in both mice and rhesus monkeys will be augmented by co-administration of plasmid-encoded chemoattractants that recruit APCs to the site of inoculation. To investigate these hypotheses, we propose: I. To investigate the immunogenicity, protective efficacy, and histopathology of a novel vaccine strategy consisting of GM-CSF/MIP-1alpha-augmented DNA vaccine priming followed by rAd5 boosting in rhesus monkeys; II. To construct plasmid chemokine/Ig fusions and to assess their ability to augment DNA vaccine-elicited immune responses in mice; III. To assess the ability of plasmid chemokines combined with plasmid costimulatory molecules to augment DNA vaccine-elicited immune responses in mice; and IV. To investigate the immunogenicity and protective efficacy of cytokine-augmented DNA vaccine priming followed by rAd5 boosting in rhesus monkeys with pre-existing anti-Ad5 immunity. The long-term objectives of these studies are to develop novel vaccine regimens that recruit APCs to the site of inoculation in animal models and to consider advancing the most promising strategies into clinical trials.